Identification of valid targets for intervention in the treatment of diabetes requires knowledge of the mechanisms of metabolic signal generation leading to stimulation of insulin secretion. Changes in Ca2+ alone cannot explain the wide dynamic range of glucose stimulated insulin secretion. We find no difference in glucose-induced Ca2+ transient between 8, 10 and 12 mM glucose where secretion increases significantly. In addition, the potent ability of the mitochondria! agonist methyl succinate (MeS) to induce secretion is not understood. Thus, we have turned our attention to additional fuel-mediated signals. Preliminary and published data document rapid concentration-dependent generation of O2-, increased cytosolic redox and decreased plasma membrane potential (??) in isolated rat islets and islet cells stimulated with MeS or glucose. Both MeS and glucose increase b-cell cytosolic redox state via increases in NADPH and glutathione (GSH). This is expected to signal redox-sensitive targets such as voltage-sensitive K+-channels (Kv), GSH and thioredoxin (TRX) networks. A role for the NADPH and O2--regulated TRX network in insulin secretion is indicated by altered secretory responsiveness in mice with a defect in this system. We hypothesize that NADPH and O2-, in the low physiological range, are additional metabolic signals that regulate a cytosolic redox network to control Kv -channels and other proteins that may play a role in such still unexplained processes as vesicular docking, Ca2+ handling or plasma membrane (PM) repolarization. The goal of the proposed work is to determine the importance of NADPH and O2- and the redox networks in physiological insulin secretion by pursuing the following aims: Aim 1. What are the mitochondrial and cytosolic metabolic signals that accompany MeS-induced insulin secretion? Studies will be done using rat islets since MeS is not a secretagogue in mouse islets and compared to mouse islets in which malic enzyme has been introduced. Aim 2: What is the role of NADPH, O2- and AT in insulin secretion in response to small increments in glucose? This aim will also evaluate the phasing of the responses. Aim 3: What are the redox mediators and their downstream targets in GSIS? This aim will evaluate the importance of changes in O2- and NADPH in regulating Kv channels. These redox sensitive channels reportedly play a role in PM repolarization. We will also determine whether NADPH, TRX or GSH are the main cytosolic redox modulators of Kv channel activity.